Methods for identifying cardiovascular therapeutic agents

ABSTRACT

The invention features screening methods which can be used to identify agents, known as vasoprotective agents, which inhibit vascular smooth muscle cell activation and/or proliferation or enhance vascular endothelial cell activation and/or proliferation or activate estrogen responsive genes in vascular cells. Preferred vasoprotective agents are relatively vasospecific, i.e., their effect on one or more types of vascular cells is more pronounced than their effect on other cell types. Treatment with such vasospecific agents will generally be associated with fewer undesirable side-effects than treatment with estrogen. 
     The methods of the invention are screening assays in which candidate agents are examined to identify vasoprotective agents. One type of screening assay involves examining the effect of a candidate agent on cell proliferation and/or cell activation. Another type of screening assay involves examining the effect of a candidate agent on the expression of a gene which is responsive to estrogen. Both screening assays involve the use of vascular cells and non-vascular cells. The use of both cell types is important because the cellular milieu is very likely to influence the effect of a candidate agent on cell proliferation, cell activation, and the expression of an given estrogen receptor responsive gene.

BACKGROUND OF THE INVENTION

Vascular diseases are the major cause of morbidity and mortality in theUnited States. Despite decades of intensive research, the mechanismsresponsible for these diseases are poorly understood.

The low incidence of vascular diseases in pre-menopausal women and therapid increase in vascular diseases, including cerebrovascular andischemic heart disease, in women following the menopause are wellrecognized. It is now recognized that the hormone estrogen plays asignificant role in preventing atherosclerotic vascular disease. Mostattempts to explain the effects of estrogen on the development ofvascular disease in women focus on indirect effects of estrogen on knownrisk factors, such as lipid and/or carbohydrate metabolism,counterbalancing of androgen-mediated effects, or indirect effects ofsex hormones on the thrombotic milieu.

Estrogen replacement therapy has been rather successful in reducing theincidence of vascular disease in post-menopausal women. It can reducethe incidence of coronary artery disease by as much as 30-50 percent.However, estrogen replacement therapy has a number of significantdrawbacks, including an increased risk of endometrial cancer, anincreased risk of breast cancer in women, and side effects such asendometrial bleeding and breast tenderness. In addition, though estrogentherapy may theoretically have beneficial effects for vascular diseasein men as well as women, attempts to examine this possibility have beenlimited by adverse effects observed with currently available agents andthe possibility of feminization that such therapy harbors.

There is a need for more selective therapies which have some or all ofthe vasoprotective benefits of estrogen therapy, but have fewerundesirable side-effects.

SUMMARY OF THE INVENTION

The present invention relates to screening methods which can be used toidentify agents which inhibit vascular smooth muscle cell activationand/or proliferation or enhance vascular endothelial cell activationand/or proliferation or activate estrogen responsive genes in vascularcells. Such agents are potentially useful for treatment or prevention ofvascular disease and are referred to as "vasoprotective agents."Preferred vasoprotective agents are relatively vasospecific, i.e., theireffect on one or more types of vascular cells is more pronounced thantheir effect on other cell types. Treatment with such vasospecificagents will generally be associated with fewer undesirable side-effectsthan treatment with estrogen. Vascular disease can result fromoverproliferation of vascular smooth muscle cells. This proliferationcan be inhibited by activation and/or proliferation of vascularendothelial cells. For example, a preferred agent would inhibit theproliferation of vascular smooth muscle cells and/or increaseproliferation of vascular endothelial cells associated with thedevelopment of atherosclerosis, but not have any significant effect oncells of the reproductive system, e.g., breast cells or uterine cells.

The methods of the invention permit identification of agents whichinhibit vascular smooth muscle cell proliferation directly by alteringthe proliferation rate of vascular smooth muscle cells. The methods ofthe invention also permit identification of agents which inhibitvascular smooth muscle cell proliferation more indirectly by enhancingthe proliferation or activation of vascular endothelial cells, whichcan, in vivo, exert an inhibitory effect on the proliferation ofvascular smooth muscle cells and can also promote vascular stabilitythrough an increased rate or extent of re-endothelialization.

The term "vasoprotective agent" refers to agents which have thepotential to reduce vascular disease in patients because they inhibit,directly or indirectly, unwanted proliferation of vascular smooth musclecells and/or enhance, directly or indirectly, endothelial cell growth orregrowth at sites of vascular injury.

The methods of the invention are screening assays in which candidateagents are examined to identify vasoprotective agents. One type ofscreening assay involves examining the effect of a candidate agent oncell proliferation and/or cell activation. Another type of screeningassay involves examining the effect of a candidate agent on theexpression of a gene which is responsive to estrogen ("an estrogenresponsive gene"). Both screening assays involve the use of vascularcells and non-vascular cells. The use of both cell types is importantbecause the cellular milieu is very likely to influence the effect of acandidate agent on cell proliferation, cell activation, and theexpression of an given estrogen receptor responsive gene.

The term "estrogen responsive gene" refers to a gene whose expressioncan be affected by the presence of an estrogen.

The methods of the invention can be used to identify agents which exerttheir effect through a known estrogen receptor, e.g., estrogen receptorα or estrogen receptor β, or through an as yet unidentified estrogenreceptor ("estrogen receptor-dependent agent"). Moreover, the methods ofthe invention can be used to identify agents which exert their effectthrough a receptor other than an estrogen receptor. These otherreceptors may recognize the same DNA binding site (recognition element)as an estrogen receptor or may bind to a recognition element which isdistinct from an estrogen receptor recognition element.

The term "estrogen receptor" means a protein which specifically binds anestrogen (e.g., 17β-estradiol or E2). Estrogen binding can be measuredusing any standard estrogen binding assay, e.g., the assay described byGibson et al. (Endocrinology, 29:2000, 1991). The term encompasses, butis not limited to, both the well-known estrogen receptor α (Green etal., Nature 320:134,1986) and the more recently described estrogenreceptor β (Kuiper et al., Proc. Nat'l Acad. Sci. USA 93:5925, 1996;Genbank Accession Number U57439).

The term "estrogen receptor-dependent agent" refers to an agent theeffect of which on a cell depends on the presence of an estrogenreceptor (e.g., estrogen receptor α or estrogen receptor β or some otherestrogen receptor) in the cell. Accordingly, the term includes agentswhich have one effect on a cell when an estrogen receptor is expressedby the cell and a different effect or no effect on the same cell when anestrogen receptor is not expressed by the cell. The agent may, ofcourse, exert other effects which are not mediated via the estrogenreceptor. Tamoxifen, 17β-estradiol, and ICI 182,780 are all examples ofestrogen receptor-dependent agents.

In selecting a vasoprotective agent, among the undesirable side-effectsto be avoided or minimized are activating or growth promoting effectson: (a) cancerous cells or pre-cancerous cells; (b) uterine cells ortissue; (c) breast cells or tissue; and (d) non-vascular,non-reproductive cells. Of course, it may not be possible to completelyavoid all or even some of these undesirable side-effects in selecting avasoprotective agent. A vasoprotective agent can be therapeuticallybeneficial even if its use is associated to some extent with some or allof these side effects.

In addition to identifying vasoprotective agents which avoid or minimizeundesirable side effects, it is desirable to identify vasoprotectiveagents which are osteoprotective. The term "osteoprotective agent"refers to agents which have the potential to reduce osteoporosis inpatients because they increase the activation and/or proliferation ofosteoclasts, which are responsible for bone formation, or because theyinhibit activation and/or proliferation of osteoclasts, which areresponsible for bone absorbtion.

Some of the methods which can be used to identify vasoprotective agentsinvolve measuring cell activation and/or proliferation. Cellproliferation can be measured by measuring cell number, DNA content, or³ H-thymidine uptake. Cell activation can be monitored by measuring theexpression of genes that are up regulated by cell activation. Forexample, to monitor activation of vascular smooth muscle cells,expression of inducible nitric oxide synthase or vascular endothelialcell growth factor can be measured. To monitor the activation ofvascular endothelial cells, expression of prostacyclin synthase orendothelial nitric oxide synthase can be measured.

Other methods which can be used to identify vasoprotective agentsinvolve measuring the expression of an estrogen responsive gene. Theeffect of a candidate agent on the expression of an estrogen receptorresponsive gene can often be most easily measured through the use of areporter construct in which the upstream regulatory region of anestrogen responsive gene (or an estrogen responsive element) is operablylinked to a readily measurable protein, e.g., luciferase. Among theestrogen responsive genes whose upstream regulatory regions can beemployed in the methods of the invention are the vitellogenin gene, theprogesterone gene, the prolactin receptor gene, the nuceleophosmin gene,and vascular endothelial cell growth factor gene.

The invention feature a method for evaluating whether an agent is avasoprotective agent, which method includes:

(a) culturing vascular endothelial cells containing a non-endogenousreporter construct in the presence of the agent, the non-endogenousreporter construct comprising an estrogen responsive element operativelylinked to a reporter gene;

(b) culturing non-vascular cells containing the non-endogenous reporterconstruct in the presence of the agent; and

(c) measuring the expression of the reporter gene in the vascularendothelial cells in the presence of the agent and measuring theexpression of the reporter gene in the non-vascular cells in thepresence of the agent;

whereby, an agent is identified as a vasoprotective agent when theexpression of the reporter gene in the vascular endothelial cells in thepresence of the agent compared to the expression of the reporter incontrol vascular endothelial cells is greater than the expression of thereporter gene in the non-vascular cells in the presence of the agentcompared to the expression of the reporter gene in control non-vascularcells.

The invention features a method for evaluating whether an agent is avasoprotective agent, which method includes:

(a) culturing vascular smooth muscle cells containing a non-endogenousreporter construct in the presence of the agent, the non-endogenousreporter construct comprising an estrogen responsive element operativelylinked to a reporter gene;

(b) culturing non-vascular cells containing the non-endogenous reporterconstruct in the presence of the agent; and

(c) measuring the expression of the reporter gene in the vascular smoothmuscle cells in the presence of the agent and measuring the expressionof the reporter gene in the non-vascular cells in the presence of theagent;

whereby, an agent is identified as a vasoprotective agent when theexpression of the reporter gene in the vascular smooth muscle cells inthe presence of the agent compared to the expression of the reporter incontrol vascular smooth muscle cells is less than the expression of thereporter gene in the non-vascular cells in the presence of the agentcompared to the expression of the reporter gene in control non-vascularcells.

The invention features a method for evaluating whether an agent is avasoprotective agent, which method includes:

(a) culturing vascular endothelial cells containing a non-endogenousreporter construct in the presence of the agent, the non-endogenousreporter construct comprising an estrogen responsive element operativelylinked to a reporter gene;

(b) culturing null cells containing the non-endogenous reporterconstruct in the presence of the agent, the null cells being cells whichdo not express an estrogen receptor; and

(c) measuring the expression of the reporter gene in the vascularendothelial cells in the presence of the agent and measuring theexpression of the reporter gene in the null cells in the presence of theagent;

whereby, an agent is identified as a vasoprotective agent when theexpression of the reporter gene in the vascular endothelial cells in thepresence of the agent compared to the expression of the reporter incontrol vascular endothelial cells is greater than the expression of thereporter gene in the null cells in the presence of the agent compared tothe expression of the reporter gene in control null cells.

The invention features a method for evaluating whether an agent is avasoprotective agent, which method includes:

(a) culturing vascular smooth muscle cells containing a non-endogenousreporter construct in the presence of the agent, the non-endogenousreporter construct comprising an estrogen responsive element operativelylinked to a reporter gene;

(b) culturing null cells containing the non-endogenous reporterconstruct in the presence of the agent, the null cells being cells whichdo not express an estrogen receptor; and

(c) measuring the expression of the reporter gene in the vascular smoothmuscle cells in the presence of the agent and measuring the expressionof the reporter gene in the null cells in the presence of the agent;

whereby, an agent is identified as a vasoprotective agent when theexpression of the reporter gene in the vascular smooth muscle cells inthe presence of the agent compared to the expression of the reporter incontrol vascular smooth muscle cells is less than the expression of thereporter gene in the null cells in the presence of the agent compared tothe expression of the reporter gene in control null cells.

The invention features a method for evaluating whether an agent is avasoprotective agent, which method includes:

(a) culturing vascular endothelial cells containing a non-endogenousreporter construct in the presence of the agent, the non-endogenousreporter construct comprising an estrogen responsive element operativelylinked to a reporter gene;

(b) culturing vascular smooth muscle cells containing the non-endogenousreporter construct in the presence of the agent; and

(c) measuring the expression of the reporter gene in the vascularendothelial cell in the presence of the agent and measuring theexpression of the reporter gene in the vascular smooth muscle cells inthe presence of the agent;

whereby, an agent is identified as a vasoprotective agent when theexpression of the reporter gene in the vascular endothelial cells in thepresence of the agent compared to the expression of the reporter incontrol vascular endothelial cells is greater than the expression of thereporter gene in the vascular smooth muscle cells in the presence of theagent compared to the expression of the reporter gene in control smoothmuscle cells.

The invention features a method for evaluating whether an agent is avasoprotective agent, which method includes:

(a) culturing vascular smooth muscle cells in the presence of the agent;

(b) culturing null cells in the presence of the agent, the null cellsbeing cells which do not express an estrogen receptor; and

(c) measuring the rate of proliferation of the vascular smooth musclecells in the presence of the agent and measuring the rate ofproliferation of the null cells in the presence of the agent;

whereby, an agent is identified as a vasoprotective agent when the rateof proliferation of the vascular smooth muscle cells in the presence ofthe agent compared to the rate of proliferation of control vascularsmooth muscle cells is less than the rate of proliferation of the nullcells in the presence of the agent compared to control null cells.

The invention features a method for evaluating whether an agent is avasoprotective agent, which method includes:

(a) culturing vascular endothelial cells in the presence of the agent;

(b) culturing null cells in the presence of the agent, the null cellsbeing cells which do not express an estrogen receptor; and

(c) measuring the rate of proliferation of the vascular endothelialcells in the presence of the agent and measuring the rate ofproliferation of the null cells in the presence of the agent;

whereby, an agent is identified as a vasoprotective agent when the rateof proliferation of the vascular endothelial cells in the presence ofthe agent compared to the rate of proliferation of control vascularendothelial cells is greater than the rate of proliferation of the nullcells in the presence of the agent compared to control null cells.

The invention features a method for evaluating whether an agent is avasoprotective agent, which method includes:

(a) culturing vascular endothelial cells in the presence of the agent;

(b) culturing vascular smooth muscle cells in the presence of the agent,the null cells being cells which do not express an estrogen receptor;and

(c) measuring the rate of proliferation of the vascular endothelialcells in the presence of the agent and measuring the rate ofproliferation of the vascular smooth muscle cells in the presence of theagent;

whereby, an agent is identified as a vasoprotective agent when the rateof proliferation of the vascular endothelial cells in the presence ofthe agent compared to the rate of proliferation of control vascularendothelial cells is greater than the rate of proliferation of thevascular smooth muscle cells in the presence of the agent compared tocontrol vascular smooth muscle cells.

The invention features a method for evaluating whether an agent is avasoprotective agent, which method includes:

(a) culturing vascular endothelial cells containing a non-endogenousreporter construct in the presence of the agent, the non-endogenousreporter construct comprising functional regulatory sequences derivedfrom a growth promoting gene operatively linked to a reporter gene;

(b) culturing non-vascular cells containing the non-endogenous reporterconstruct in the presence of the agent; and

(c) measuring the expression of the reporter gene in the vascularendothelial cells in the presence of the agent and measuring theexpression of the reporter gene in the non-vascular cells in thepresence of the agent;

whereby, an agent is identified as a vasoprotective agent when theexpression of the reporter gene in the vascular endothelial cells in thepresence of the agent compared to the expression of the reporter incontrol vascular endothelial cells is greater than the expression of thereporter gene in the vascular smooth muscle cells in the presence of theagent compared to the expression of the reporter gene in control smoothmuscle cells.

The invention features a method for evaluating whether an agent is avasoprotective agent, which method includes:

(a) culturing vascular smooth muscle cells containing a non-endogenousreporter construct in the presence of the agent, the non-endogenousreporter construct comprising functional regulatory sequences derivedfrom a growth promoting gene operatively linked to a reporter gene;

(b) culturing null cells containing the non-endogenous reporterconstruct in the presence of the agent, the null cells being cells whichdo not express an estrogen receptor; and

(c) measuring the expression of the reporter gene in the vascular smoothmuscle cells in the presence of the agent and measuring the expressionof the reporter gene in the non-vascular cells in the presence of theagent;

whereby, an agent is identified as a vasoprotective agent when theexpression of the reporter gene in the vascular smooth muscle cells inthe presence of the agent compared to the expression of the reporter incontrol vascular smooth muscle cells is less than the expression of thereporter gene in the non-vascular cells in the presence of the agentcompared to the expression of the reporter gene in control non-vascularcells.

The invention features a method for evaluating whether an agent is avasoprotective agent, which method includes:

(a) culturing vascular endothelial cells containing a non-endogenousreporter construct in the presence and absence of the agent, thenon-endogenous reporter construct comprising functional regulatorysequences derived from a growth promoting gene operatively linked to areporter gene;

(b) culturing vascular smooth muscle cells containing the non-endogenousreporter construct in the presence and absence of the agent; and

(c) measuring the expression of the reporter gene in the vascularendothelial cells in the presence of the agent and measuring theexpression of the reporter gene in the vascular smooth muscle cells inthe presence of the agent;

whereby, an agent is identified as a vasoprotective agent when theexpression of the reporter gene in the vascular endothelial cells in thepresence of the agent compared to the expression of the reporter incontrol vascular endothelial cells is greater than the expression of thereporter gene in the vascular smooth muscle cells in the presence of theagent compared to the expression of the reporter gene in controlvascular smooth muscle cells.

The invention features a method for evaluating whether an agent is avasoprotective agent, which method includes:

(a) culturing vascular endothelial cells containing a non-endogenousreporter construct in the presence of the agent, the non-endogenousreporter construct comprising functional regulatory sequences derivedfrom a growth arrest gene operatively linked to a reporter gene;

(b) culturing non-vascular cells containing the non-endogenous reporterconstruct in the presence of the agent; and

(c) measuring the expression of the reporter gene in the vascularendothelial cells in the presence of the agent and measuring theexpression of the reporter gene in the non-vascular cells in thepresence of the agent;

whereby, an agent is identified as a vasoprotective agent when theexpression of the reporter gene in the vascular endothelial cells in thepresence of the agent compared to the expression of the reporter incontrol vascular endothelial cells is less than the expression of thereporter gene in the vascular smooth muscle cells in the presence of theagent compared to the expression of the reporter gene in control smoothmuscle cells.

The invention features a method for evaluating whether an agent is avasoprotective agent, which method includes:

(a) culturing vascular smooth muscle cells containing a non-endogenousreporter construct in the presence of the agent, the non-endogenousreporter construct comprising functional regulatory sequences derivedfrom a growth arrest gene operatively linked to a reporter gene;

(b) culturing null cells containing the non-endogenous reporterconstruct in the presence of the agent, the null cells being cells whichdo not express an estrogen receptor; and

(c) measuring the expression of the reporter gene in the vascular smoothmuscle cells in the presence of the agent and measuring the expressionof the reporter gene in the non-vascular cells in the presence of theagent;

whereby, an agent is identified as a vasoprotective agent when theexpression of the reporter gene in the vascular smooth muscle cells inthe presence of the agent compared to the expression of the reporter incontrol vascular smooth muscle cells is greater than the expression ofthe reporter gene in the non-vascular cells in the presence of the agentcompared to the expression of the reporter gene in control non-vascularcells.

The invention features a method for evaluating whether an agent is avasoprotective agent, which method includes:

(a) culturing vascular endothelial cells containing a non-endogenousreporter construct in the presence and absence of the agent, thenon-endogenous reporter construct comprising functional regulatorysequences derived from a growth arrest gene operatively linked to areporter gene;

(b) culturing vascular smooth muscle cells containing the non-endogenousreporter construct in the presence and absence of the agent; and

(c) measuring the expression of the reporter gene in the vascularendothelial cells in the presence of the agent and measuring theexpression of the reporter gene in the vascular smooth muscle cells inthe presence of the agent;

whereby, an agent is identified as a vasoprotective agent when theexpression of the reporter gene in the vascular endothelial cells in thepresence of the agent compared to the expression of the reporter incontrol vascular endothelial cells is less than the expression of thereporter gene in the vascular smooth muscle cells in the presence of theagent compared to the expression of the reporter gene in controlvascular smooth muscle cells.

In each of the assays described herein the expression a reporter gene ina cell grown in the presence of an agent is always corrected bycomparison to the expression of a reporter gene in an otherwiseidentical cell grown in the absence of the agent.

The term "estrogen responsive element" refers to a DNA sequence elementwhich confers upon a gene to which it is operably linked responsivenessto the presence of an estrogen. The so-called consensus estrogenreceptor α response element, is an example of an estrogen responsiveelement. Estrogen responsive elements can be found in the upstreamregulatory region of estrogen reponsive genes, e.g., the human VEGF geneor the vitellogenin gene. While the entire upstream regulatory region ofan estrogen responsive gene can be used as an estrogen responsiveelement, it is also possible to identify one or more smaller estrogenresponsive elements within the upstream regulatory region. This isbecause an estrogen responsive element includes the smallest unit whichconfers estrogen responsiveness.

A "reporter gene" is a sequence which includes a mammalian promoter, asequence encoding a detectable protein, e.g., luciferase, and, in mostcases, a poly(A) signal sequence. Reporter genes are routinely used toinvestigate upstream regulatory regions. Generally, all or a portion ofan upstream regulatory region of interest is position upstream (5') ofthe reporter gene promoter and the entire construct is introduced into aselected cell type to study the effect of the selected upstream regionon expression of the protein encoded by the reporter gene. Reportergenes are often inserted into vectors. For example, the pGL3-PromoterVector (Promega, Madison, Wis.) is a vector which contains an SV40promoter upsteam of a firefly luciferase gene (including a polyA signalsequence). The vector includes sequences required for replication of thevector in E. coli and mammalian cells.

A sequence element is "operably linked" to a reporter gene when it isinserted upstream of the reporter gene promoter such that can influenceexpression of the protein encoded by the gene. For example, an estrogenresponsive element inserted into the polylinker found upstream of theSV40 promoter in pGL3-Promoter Vector is operably linked to theluciferase reporter gene.

In various preferred embodiments the vascular, non-vascular, or nullcells are stably transfected with the non-endogenous reporter construct;the vascular, non-vascular, or null cells are co-transfected with anestrogen receptor expression construct. In more preferred embodimentsthe estrogen receptor expression construct expresses estrogen receptor αand the estrogen receptor expression construct expresses estrogenreceptor β.

In another aspect the invention features a vascular endothelial cellcontaining an estrogen receptor β expression construct. In yet anotheraspect the invention features a vascular smooth muscle cell containingan estrogen receptor β expression construct.

In another aspect the invention features cells of cell line WB572. Inpreferred embodiments the WB572 cells contain a non-endogenous reporterconstruct comprising an estrogen responsive element operatively linkedto a reporter gene, contain an estrogen receptor α expression construct,and contain an estrogen receptor β expression construct. In morepreferred embodiments the estrogen receptor α expression construct is aninducible expression construct and the estrogen receptor β expressionconstruct is an inducible expression construct.

In all assays entailing the measurement and comparison of cellproliferation rates the measured rate of proliferation of a given celltype in the presence of an agent is always corrected for the rate ofproliferation of a control cell. The control cell is an otherwiseidentical cell grown the absence of the agent being tested.

When measuring and comparing proliferation rates it should be understoodthat some vasoprotective agents may actually reduce the rate ofproliferation of certain cell types. For example, a desirablevasoprotect agent might not substantially change the rate ofproliferation of a selected strain of vascular endothelial cell whiledecreasing the rate of proliferation of a selected strain of vascularsmooth muscle cells.

In various preferred embodiments the vascular, non-vascular, or nullcells are stably transfected with the non-endogenous reporter construct;the vascular, non-vascular, or null cells are co-transfected with anestrogen receptor expression construct. In more preferred embodimentsthe estrogen receptor expression construct expresses estrogen receptor αand the estrogen receptor expression construct expresses estrogenreceptor β.

DETAILED DESCRIPTION

Described below are various methods for screening candidate agents toidentify vasoprotective agents.

The methods of the invention rely on examining the effect of variousagents based on either its effect on the growth and/or activation ofparticular cell types (e.g., vascular and non-vascular) or its effect onthe expression of an estrogen responsive gene in particular cell types.The cellular context is critical because the expression of a gene underthe control of an estrogen receptor may also be affected by one or moreadditional factors, such as co-activators (e.g., SRC-1), repressors(e.g., NCOR/SMRT), or other expression modulating proteins which may becell specific. Cell specific factors include those factors which may ormay not be expressed in a given cell type or which may be expressed atdifferent levels in different cell types. Although these additionalfactors can contribute to the cell-type specific nature of geneactivation by asteroid hormone receptor such as the estrogen receptor,the methods of the invention do not require an understanding of theseadditional factors.

A vasoprotective agent can be identified based on either its effect onthe growth and/or activation of particular cell types (e.g., vascularand non-vascular) or its effect on the expression of an estrogenresponsive gene in particular cell types (e.g., vascular andnon-vascular).

The effect of a given agent on cell growth and/or activation can bemeasured either directly or indirectly. Direct measurement of cellgrowth and/or activation involves monitoring cell proliferation oractivation. Indirect measurement of cell growth and/or activationinvolves monitoring the expression of genes encoding cell growthpromoting factors and/or monitoring the expression of genes encodingcell growth arrest factors. In general, the expression of these genes ismonitored indirectly by monitoring the expression of a reporterconstruct in which all or part of the upstream regulatory region of thegene of interest is operably linked to a sequence encoding a readilydetectable protein.

As noted above, a vasoprotective agent can also be identified based onits effect on the expression of an estrogen responsive gene inparticular cell types. An estrogen responsive gene can be anaturally-occurring gene which is responsive to estrogen, e.g., thevascular endothelial cell growth factor gene. The effect of an agent onthe expression of such a naturally-occurring estrogen-responsive genecan be measured directly or indirectly. Direct measurement involvesmonitoring the expression of the estrogen responsive gene itself. Onecan measure the expression of a genomic copy of the gene or one canmeasure the expression of a non-endogenous copy of the gene, e.g., acopy introduced by transfection. Generally, expression of the estrogenresponsive gene is monitored by measuring mRNA production.Alternatively, protein production can be measured. In many cases it ispreferable to indirectly monitor the expression of an estrogenresponsive gene by monitoring the expression of a reporter construct inwhich all or part of the upstream regulatory region of the estrogenresponsive gene of interest is operably linked to a sequence encoding areadily detectable protein. Another type of estrogen responsive gene isa wholly synthetic gene in which sequences known to confer estrogenresponsiveness are operably linked to a sequence encoding a readilydetectable protein. A luciferase encoding reporter construct in whichluciferase expression is under the control of a consensus estrogenreceptor α recognition element is an example of such an estrogenresponsive gene.

Cells

As discussed above, the methods of the invention identify an agent as acandidate vasoprotective agents by examining the effect of the agent oncell growth and/or activation or gene expression in two differentcellular contexts: vascular and non-vascular. Accordingly, the methodsof the invention can employ a variety of cell types.

Vascular cell lines of particular interest are: WB572 cells(spontaneously transformed human saphenous vein smooth muscle cells) andSV-E6 cells (human saphenous vein cells stably transfected with the E6viral oncogene).

Other vascular cell lines useful in the method of the invention include,but are not limited to: A7R5 cells (spontaneously transformed ratthoratic aorta smooth muscle cells; ATCC, Bethesda, Md.; ECV304 cells(human umbilical vein vascular endothelial cells; ATCC, Bethesda, Md.);GH3B6 cells (rat pituitary cells; ATCC, Bethesda, Md.); PVEC cells (ratpulmonary vein endothelial cells; J. Tissue Culture Res. 10:9, 1986);CPA47 (bovine endothelial cells; ATCC CRL 1733); CPAE cells (bovineendothelial cells; ATCC CCL 209); EJG cells (bovine endothelial cells;ATCC CRL 8659); FBHE (bovine endothelial cells; ATCC CRL 1395); HUV-EC-Ccells (human endothelial cells; ATCC CRL 1730); and T/G HA-VSMC cells(human vascular smooth muscle cells; ATCC CRL 1999).

Non-vascular cell lines useful in the methods of the invention include,but are not limited to: MCF-7 cells (human breast cancer cells; ATCC,Bethesda, Md.); ZR-75-1 cells (human breast cancer; ATCC CRL-1500);UACC-893 cells (human breast cancer; ATCC CRL-1902); RL95-2 cells (humanendometrial cancer; ATCC CRL-1671); KLE cells (human endometrial cancer;ATCC CRL-1622).

Cell lines which do not express estrogen receptor α, e.g., COS-1 cells,Cos-7 cells, CHO cells, HEK 293 cells, and HeLa cells, are useful insome embodiments of the invention.

Bone cell lines, e.g., UMR-106 cells (rat osteogenic sarcoma; ATCCCRL-1661) and HOS (human osteosarcoma; ATCC CRL-1543), are useful fordetermining whether a candidate vasoprotective agent is alsoosteoprotective.

Reporter Constructs

Reporter constructs are used to indirectly monitor the effect of anagent on the proliferation and/or activation of vascular cells and tomonitor the effect of an agent on the expression of an estrogenresponsive gene.

Reporters Based on Estrogen Responsive Genes

A number of reporter constructs in which all or a portion of theupstream regulatory region of an estrogen receptor responsive gene or anisolated estrogen receptor recognition element (ERE) is operably linkedto a sequence encoding a detectable protein are useful in the methods ofthe invention.

In general, any gene which is responsive to an estrogen receptor canserve as the basis for a reporter construct. For example, the followingvascular genes are potentially of interest: prostaglandincyclooxygenase, prostaglandin synthase, nitric oxide synthase(constitutive or calcium-dependent), collagen, elastin, c-fos,progesterone receptor, vascular endothelial growth factor, epidermalgrowth factor receptor, interleukin-6, neu, egr-1, estrogen receptor,heat shock protein 27, vascular adhesion molecules, vascular smoothmuscle cell calcium channels, ryanodine receptor, FLT4 receptor tyrosinekinase, fibroblast growth factor receptor, and inducible nitric oxidesynthase. Other potentially estrogen responsive genes are described inMendelsohn and Karas (Current Opinion in Cardiology 9:619, 1994).

In some cases an estrogen responsive gene will also be a growth relatedgene. In these cases it the change is expression indicative of avasoprotective agent may depend on the cell type. Thus, in the followinglist a "+" indicates that preferred agents increase expression of thatgene (or a reporter operably linked to the upstream control region ofthat gene in the indicated cell type; a "-" indicates that preferredagents decrease expression of that gene (or a reporter operably linkedto the upstream control region of that gene in the indicated cell type.In each case the format is (preferred response in vascular endothelialcells/preferred response in vascular smooth muscle cells): prostaglandincyclooxygenase (+/+), prostaglandin synthase (+/+), nitric oxidesynthase (constitutive or calcium-dependent) (+/+), collagen (-/-),elastin (-/-), c-fos (+/-), progesterone receptor (+/+), vascularendothelial growth factor (+/+), epidermal growth factor receptor (-/-),interleukin-6 (+/+), neu (-/-), egr-1 (-/-), estrogen receptor (+/+),heat shock protein 27 (+/-), vascular adhesion molecules (-/-), vascularsmooth muscle cell calcium channels (-/-), ryanodine receptor (-/-),FLT4 receptor tyrosine kinase (+/-), fibroblast growth factor receptor(-/-), and inducible nitric oxide synthase (+/+).

All or part of the upstream regulatory region of a naturally-occurringestrogen responsive gene can be operably linked to the sequence encodinga detectable protein in order to provide a suitable reporter construct.The portion of the upstream regulatory region used must include asequence which confers some degree of responsiveness to estrogen.

One useful reporter construct is pmVEGF-Luc. In this reporter construct,a 1.6 kB fragment of genomic DNA located upstream of the murine vascularendothelial cell growth factor (VEGF) gene has been inserted into thepGL2-Basic luciferase reporter plasmid (Promega; Madison, WI). The 1.6kB fragment includes the transcription start site, 1.2 kB of sequenceupstream of the start site, and 0.4 kB of sequence downstream of thestart site. Useful variants of this reporter plasmid can be made bydeleting a portion of the murine VEGF control region. For example,pVEGF-Luc-Apa/Kpn is identical to pVEGF-luc except that 455 bp at the 5'end of the 1.6 kB fragment have been removed; pVEGF-Luc-Mlu is identicalto pVEGF-luc except that 768 bp at the 5' end of the 1.6 kB fragmenthave been removed; and pVEGF-Luc-Sma is identical to pVEGF-luc exceptthat 455 bp at the 5' end of the 1.6 kB fragment have been removed.Similar reporter constructs can be created using upstream regulatorysequences from the human VEGF gene. For example, phVEGF-Luc is areporter construct in which 5.1 kb genomic DNA upstream of the humanVEGF coding sequence inserted into the pGL2-Basic luciferase reporterplasmid. This particular construct includes 3.4 kb of sequence upstreamof the VEGF transcription start site and 1.7 kb untranslated sequencedownstream of the transcription start site.

Assays Based on Estrogen Responsive Reporters

Agents which activate the expression of estrogen responsive genes invascular cells, but not in non-vascular cells are candidatevasoprotective agents. Generally, reporter constructs can be used toexamine the effect of a given agent on the expression of such genes.Suitable reporter constructs can be created by placing a sequenceencoding a readily detectable protein, e.g., luciferase, under thecontrol of all or part the upstream transcription control region of anestrogen responsive gene. The portion of the upstream control regionused in the reporter construct must include the recognition element(s)required for response to estrogen. Such elements can be identified basedon their similarity to known estrogen receptor recognition elements orby functional dissection of the upstream regulatory region.

Alternatively, a reporter gene can be placed under the control of one ormore synthetic estrogen receptor response elements. Such syntheticelements can be designed based on the sequence of known estrogenreceptor response elements. Both types of constructs are referred to asestrogen responsive reporters.

To set up the assay, a selected estrogen responsive reporter isintroduced into a vascular cell line (a vascular endothelial cell lineor a vascular smooth muscle cell line) and a non-vascular cell line. Thenon-vascular cell line is preferably a cell line derived from areproductive tissue (e.g., a breast cell line or a uterine cell line).The selected estrogen responsive reporter may also be introduced into acell line which does not express estrogen receptor α or β (null cells).Suitable null cell lines include COS cells, HeLa cells, and HEK 293cells. While the reporter can be introduced into the cells various celllines by transient transfection, it is preferable that the cells bestably transfected with the reporter construct. Generally, human,murine, and/or rat cells can be used in the methods of the invention. Inaddition, the reporter constructs can employ the upstream regulatorysequences of human, murine, or rat estrogen responsive genes. Moreover,the cell line and the gene used as the source of upstream regulatoryregions for the reporter construct do not have to be of the samespecies. Thus, a reporter in which the upstream control region of murineVEGF gene is operably linked to sequence encoding luciferase can be usedin the screening methods of the invention.

There are three preferred formats for a screening assay which is basedon an estrogen responsive reporter. All three formats involve at leasttwo different cell types which harbor the same estrogen responsivereporter. The preferred assays use either vascular cells andnon-vascular cells or vascular cells and null cells or two differenttypes of vascular cells. However, other combinations may be used.Moreover, the vascular/non-vascular format and the vascular/vascularformat can employ more than one vascular cell type (e.g., a vascularsmooth muscle cell line and a vascular endothelial cell line or twodifferent vascular endothelial cell lines) and more than onenon-vascular cell type (e.g., a breast cell line and a uterine cellline). The use of two or more different cell lines of the same type in asingle assay format is desirable because one can identify candidatevasoprotective agents which have the same or similar effect on severalcells of the same type. The vascular/null format can also employ morethan one cell line of each type. In both formats one can use two or moredifferent estrogen responsive reporter constructs.

EXAMPLE Vascular Cell/Non-vascular Cell Format

The following example is meant to be illustrative, not limiting. Forillustrative purposes, this example employs cells which have been stablytransfected with a luciferase reporter construct. The assay, asdescribed, is configured for cells grown in 96 well plates. The assay isnot in any way confined to these characteristics and can equally wellemploy transiently transfected cells, reporter proteins other thanluciferase, or cells grown in tissue culture wells of other sizes. Theseparameters were chosen as examples because they afford the considerableopportunity for automation and high throughput.

Cells are plated in 96 well plates in complete medium at 70% confluence.Each assay, which can accommodate 8 different cell lines, includes cellsof vascular and non-vascular origin. After plating, the cells areallowed to rest undisturbed for 6 hours to allow attachment, and theneach well is rinsed twice with 200 μl of serum free media and thenre-fed with 200 μl of serum-free media that contains a candidate agentof interest. Each cell type is exposed to three different concentrationsof the compound of interest and to control solution without thecandidate agent. Each treatment is performed in triplicate. Thus, asconfigured here, each assay includes triplicate measurements of 8 celllines under four different experimental conditions for a total of 96samples. After 48 hours of incubation, the cells are lysed in situ in 50μl of a solution compatible with the luciferase assay (Karas et al.,Circulation 89:1943, 1994) and luciferase activity is determinedspectrophotometrically (either manually by removal of the cell lysatefrom the tissue culture plate, or automatically in a computerized platereader). Agents that increase the luciferase activity over controlconditions in the vascular cells are identified as vasoactive agents.Agents that are vasoactive, but do not alter luciferase activity in thenon-vascular are identified as candidate vasoprotective agents.

The general assay described above is amenable to variation in a numberof ways. As noted above, the assay can be employ transiently transfectedcells or stably transfected cells. The use of transiently transfectedcells is particularly well suited to rapid testing of new cell lines todetermine their suitability for further study. In some cell types, thelevel of activation of the estrogen responsive reporter construct may below, and this may be augmentedtra co-transfection (again in either atransient or a stable fashion) of an estrogen receptor (or an isoform ormutant thereof) expression construct. In addition, the level ofexpression of the estrogen receptor expression construct can becontrolled by use of a regulatable promotor such as thetetracycline-responsive system described below. Other means ofregulating the estrogen receptor expression construct are alsocompatible with this screening assay.

EXAMPLE Vascular Cell/Null Cell Format

The following example is for illustrative purposes only. A vascularsmooth muscle cell line, WB572 cells, and a null cell line, COS cells,are stably transfected with the estrogen responsive reporter pERE-Luc.The cells are grown and treated as described above. Any increase inluciferase expression of treated WB572 cells compared to control cellsis compared to the increase in luciferase expression of treated COScells compared to control cells. An agent which increases luciferaseexpression in WB572 cells to a greater extent than COS cells is acandidate vasoprotective agent.

EXAMPLE Vascular Cell/Vascular Cell Format

A vascular smooth muscle cell line and a vascular endothelial cell lineare stably transfected with the estrogen responsive reporter pERE-Luc.The cells are grown and treated as described above. Agents whichincrease luciferase expression in both cell types are candidatevasoprotective agents.

Reporters Based on Growth-Promoting and Growth-Arrest Genes

Agents which cause vascular endothelial cells to proliferate or becomeactivated without causing significant proliferation or activation ofvascular smooth muscle cells or non-vascular cells or which causeinhibition of vascular smooth muscle cell proliferation are alsocandidate vasoprotective agents. Cell proliferation or activation can bemeasured directly by any conventional method, e.g., by monitoring ³H-thymidine uptake. In addition, cell proliferation and/or cellactivation can be measured indirectly by measuring the expression of areporter gene under the control of the upstream regulatory region of agrowth-related gene. The growth related gene can be a gene whoseexpression is associated with growth, e.g., AP1 (fos/jun), c-myc, orc-myb or growth inhibition or arrest, e.g., p21, p27, p53, gas, gax, orRb.

All or part of the upstream regulatory region of such a growth-relatedgene can be operably linked to the sequence encoding a detectableprotein in order to provide a suitable reporter construct.

Assays Based on Growth Promoting Related Reporters or Growth ArrestRelated Reporters

A potential vasoprotective agent is one which increases the expressionof a growth related gene in vascular endothelial cells, but whichinhibits or does not significantly increase the expression of the samegrowth promoting related gene in vascular smooth muscle cells and/ornon-vascular cells. An agent which increases the expression of a growtharrest related genes in vascular smooth muscle cells, but does notsignificantly increase the expression of the same growth arrest relatedgene in vascular endothelial cells is also a candidate vasoprotectiveagent.

Screening assays based on growth promoting related reporters or growtharrest related reporters can employ either of two preferred formats.Both formats involve at least three different cell types which harborthe same growth related reporter or growth arrest related reporter. Thepreferred assays use either a vascular cell line and a non-vascular cellline or a vascular cell line and a null cell line or a vascularendothelial cell line and a vascular smooth muscle cell line. However,other combinations may be used. Moreover, each format can employ morethan one cell line of each type. The use of two or more different celllines of the same type is desirable because, when the related cell linesexhibit the same response to a given candidate agent, one can be morecertain that the effect of the agent will be similar for all cells ofthat type.

Agents Useful in Screening Assays

Agents for use in the screening assays of the invention can be obtainedfrom any source. Libraries of synthetic and/or natural compounds areparticularly useful. Numerous means are currently used for random anddirected synthesis of saccharide, peptide, nucleic acid, and smallmolecule compounds. Synthetic compound libraries are commerciallyavailable from Maybridge Chemical Co. (Trevillet, Cornwall, UK),Comgenex (Princeton, N.J.), Brandon Associates (Merrimack, N.H.), andMicrosource (New Milford, Conn.). A rare chemical library is availablefrom Aldrich (Milwaukee, Wis.). Libraries of natural compounds in theform of bacterial, fungal, plant and animal extracts are available fromPan Laboratories (Bothell, Wash.) and MycoSearch (NC). Phytoestrogens(Maleka et al., Environ. Health Perspectives 102:572, 1994; Knight etal., Maturitas 22:167, 1995) and the components of Premarin (Stern,Maturitas 4:33, 1982; Jayalilaka et al., J. Organic Chrom. 617:19, 1993)can also be screened using the methods of the invention.

Augmentation of Estrogen Receptor Expression

Under some circumstances, e.g., when the signal from the growth relatedreporter or the growth arrest related reporter is relatively low, it maybe desirable to transfect the cells with an estrogen receptor expressionconstruct in addition to the reporter construct. The estrogen receptorexpression construct can express estrogen receptor α or estrogenreceptor β. Under some circumstances it may be desirable to introduceboth an estrogen receptor α expression construct and an estrogenreceptor β expression construct.

Estrogen Receptors

Estrogen receptor α is a transcription factor which belongs to thenuclear receptor superfamily. Like other members of the superfamily,estrogen receptor α, upon activation, e.g., by binding of its ligand,binds to DNA and alters gene expression. When estrogen receptor iscomplexed with its ligand, estrogen, it binds specific DNA sequences, aswithin the regulatory regions of a number of target genes. Whether thebound receptor has a positive or negative effect on the transcription ofa given target gene, depends on both the cell type and the context ofthe regulatory region (Tzukerman et al., Mol. Endocrin. 8:21, 1994). Thegene encoding estrogen receptor α has been cloned and shown to befunctional in heterologous cells, including vascular cells, into whichit has been introduced (Kumar et al., EMBO J. 5:2231, 1986; Green etal., Science 231:1150, 1986; Karas et al., Circulation 89:1943, 1994).

Estrogen receptor α is thought to include at least two importanttranscription activation domains. Binding of estrogen activates one ofthese domains, the transcription-activation factor-2 (TAF-2) domain.Activation of the other domain, the transcription-activation factor-1(TAF-1) domain, is not dependent on estrogen binding, but may influenceTAF-2 mediated transactivation and may be activated by other mechanismssuch as phosphorylation via growth factor-mediated pathways.

Estrogen receptor β (Kuiper et al., Proc. Nat'l Acad. Sci. USA 93:5925,1996; Genbank Accession Number U57439) is another estrogen receptorwhich may mediate the effects of estrogen on vascular cells. Ratestrogen receptor β, in the presence of estrogen, increases expressionof a reporter gene under the control of a vitellogenin promoter estrogenresponse element.

Variant and Mutant Estrogen Receptors

In addition to wild-type estrogen receptors α and β, a number of variantestrogen receptors may be employed in the screening methods describedbelow. Of particular interest are variant or mutant estrogen receptorwhich permit one to discriminate TAF-1 mediated effects and TAF-2mediated effects. The variant estrogen receptors ESTROGEN RECEPTOR-S118Aand ESTROGEN RECEPTOR-TAF2 are examples of estrogen receptors useful insome embodiments of the invention. The first of these mutants, ESTROGENRECEPTOR-S118A, has a point mutation at amino acid 118. This mutationcreates a molecule in which the TAF1 domain cannot be activated byphosphorylation of Ser 118. The second of these mutants, ESTROGENRECEPTOR-TAF-2, has three mutations (at amino acids 538, 542, and 545)which together inactivate the TAF-2 domain so that the TAF-1 domain isthe primary functional activation domain.

Production of Estrogen Receptors and Estrogen Receptor Variants

Any suitable expression construct can be used to express an estrogenreceptor or an estrogen receptor variant. Estrogen receptor expressionconstructs can be used to express an estrogen receptor or an estrogenreceptor variant in both cells which have an endogenous estrogenreceptor and those which do not. For example, pCMV3-ESTROGEN RECEPTOR,plasmid which expresses estrogen receptor α expression construct wascreated by cloning the wild-type, human estrogen receptor e cDNA EcoRIfragment (Tora et al., EMBO J. 8:1981, 1989) into pCDNA3.1 (Invitrogen).Constructs for expression of variant estrogen receptors, pCMV3-ESTROGENRECEPTOR-S106A (coding for an alanine instead of a serine at amino acidposition 106), pCMV3ER-S118A (coding for an alanine rather than a serineat position 118), pCMV3-ESTROGEN RECEPTOR-283 (in which a terminationcodon was introduced following amino acid 283), and pCMV3-ESTROGENRECEPTOR-TAF2mut (in which amino acids 538, 542, and 545 where allchanged to alanine), were constructed by site directed mutagenesis(Kunkel et al., Proc. Nat'l Acad. Sci. USA 82:488, 1985). A plasmidencoding a truncated version of the estrogen receptor containing onlythe A/B and C domains, pCMV3-ESTROGEN RECEPTOR-271, was constructed byexcising from pCMV3-ESTROGEN RECEPTOR the coding sequence from the XcmIsite at bp 815 to the end of the coding sequence.

A series of inducible estrogen receptor α expression constructs werecreated by cloning sequences encoding wild-type and variant estrogenreceptor α into pUHD10-3 (Mol. Cell. Biol., 4:1669, 1994). This plasmidused in conjunction with either pUHD 15-1neo (Mol. Cell. Biol., 4:1669,1994) or pUHD172-1neo (Science 268:1766, 1995) allow fortetracycline-responsive control of estrogen receptor expression. Theseadditional estrogen receptor expression plasmids are referred to as:pTet-ESTROGEN RECEPTOR, pTet-ESTROGEN RECEPTOR-S106A, and pTet-ESTROGENRECEPTOR-S118A. These inducible estrogen receptor expression constructsare useful for determining whether an observed effect on a cell isdependent on the presence of the estrogen receptor.

Expression constructs, including inducible constructs, for theexpression of estrogen receptor β can be prepared as described above.DNA encoding estrogen receptor β can be obtained as described in Kuiperet al. (Proc. Nat'l Acad. Sci. USA 93:5925, 1996) or can be isolated byPCR cloning using primers based on sequences disclosed in Kuiper et al.(Proc. Nat'l Acad. Sci. USA 93:5925, 1996).

Detectable Proteins

The reporter constructs can employ any suitable detectable protein,e.g., luciferase, green flourscent protein, or chloramphenicol acetyltransferase (CAT).

Vascular Endothelial Cell and Vascular Smooth Muscle Cell Co-CulturingMethods

Both the assays based on estrogen responsive reporter and the assaysbased in growth related or growth arrest related reporters can beperformed using a co-culturing format. In a co-culturing assay anyvascular endothelial cell line is grown in co-culture with a vascularsmooth muscle cell line and any vascular smooth muscle cell line isgrown in co-culture with a vascular endothelial cell line. Co-culturingcan be accomplished by actually growing a mixed cell culture or by usinga Transwell® cell culture insert (Costar Corp.). The second vascularcell line does not contain a reporter construct; it is present toprovide the first vascular cell line (i.e., the reporter harboring cellline) with an environment which more closely resembles that found invivo.

In vivo Model for Screening Vasoprotective Agents

Vasoprotective agents identified using the screening methods describedherein can be tested in various animal models of vascular injury. Forexample, agents identified using the method described herein can betested in the mouse cartoid artery injury model described by Linder etal. (Circ. Res. 73:792, 1993). Agents may also be tested using theporcine femoral injury model. (Nabel et al., Science 249:1285, 1990;Nabel et al., Nature 362:844, 1993; and Ohno et al., Science 265:781,1994).

Induction of Estrogen Responsive Genes by Estrogen

To study the role of estrogen on expression of the VEGF gene in vascularsmooth muscle cells, human saphenous vein smooth muscle cells (HSVSMC)and HeLa cells were transiently transfected with a VEGF-luciferasereporter construct. In this reporter construct, expression of luciferaseis under the control of a 1.2 kB fragment of the murine VEGF generegulatory region. The cells were also transfected with an estrogenreceptor α expression construct. All of the results described below havebeen normalized for transfection efficiency.

The luciferase expression of VEGF-luciferase transfected HSVSMC wasincreased 1.7±0.2 fold (n=12, p<0.02) when the cells were grown in thepresence of 1 μM estrogen. To confirm that this activation of luciferaseexpression was estrogen receptor α dependent, HeLa cells, which do notposses endogenous estrogen receptor α, were also studied. Growth ofVEGF-luciferase reporter transfected HeLa cells in estrogen containingmedia had no effect on luciferase expression. However, if the cells wereco-transfected with an estrogen receptor α expression plasmid, growth inestrogen containing media increased luciferase expression 4.2±0.8-fold(n=21, p<0.03).

Estrogen Activates Vascular Endothelial Growth Factor Expression inHuman Vascular Cells but not in Non-vascular Cells.

The following experiments demonstrate that estrogen activates VEGFexpression in vascular cells (WB572 cells), but not in non-vascularcells such as rat pituitary cells (GH3B6). In these experiments WB572cells and GHB6 cells were transiently transfected with the pmVEGF-Lucreporter construct either with or without an expression plasmid forestrogen receptor α. The cells were cultured for 48 hours in the absenceor presence of estrogen (10⁻⁶ M) and luciferase expression was measured.Estrogen induced a 1.8-fold increase in luciferase activity in WB572cells, but had no effect in GH3B6 cells. The ability of the GH3B6 cellsto respond to estrogen was confirmed by transfection of the pERE-Lucreporter which was activated 8-fold under the same conditions.Interestingly, similar experiments performed with rat VSMC (A7r5 cells)demonstrated that estrogen did not activate VEGF expression in thesecells either. Thus, the effects of estrogen on gene expression candepend both on the type of cells studied as well as on the species fromwhich the cell is derived.

Mitogen-mediated (Ligand Idependent) Activation of the Estrogen Receptorin Vascular Cells is Cell-type Dependent

The following experiments demonstrate that the estrogen receptor isactivated in cells by mitogenic stimulation with epidermal growth factor(EGF), but that this activation is dependent on the type of cellstudied. In these experiments three varieties of vascular cells (PVEC,HSVSMC, and human aortic VSMC) and the non-vascular cell-type HeLacells, were transiently transfected with the reporter plasmid ERE-Lucand an expression plasmid for the estrogen receptor. The cells were thencultured for 48 hours in serum-free medium in the absence or presence ofEGF (100 ng/ml). The degree to which the estrogen receptor is able toactivate the ERE is then determined by measuring luciferase activity incell lysates. In all three types of vascular cells, EGF inducedapproximately a 3-fold increase in luciferase activity. In HeLa cells,EGF had no effect. The cell-type specificity of the EGF effect wasdemonstrated in these same experiments by showing (a) either estrogen orFBS induced similar increases in receptor activation in all cell typesstudied, and (b) PDGF did not activate the estrogen receptor in any celltype studied.

Estrogen Inhibition of Medial Vascular Smooth Muscle Cell ProliferationOccurs via a Pathway Which is Not Dependent on Estrogen Receptor α

The experiments described below demonstrate that estrogen inhibition ofmedial vascular smooth muscle cell proliferation is mediated,principally, by a estrogen receptor α-independent pathway.

The effects of estrogen on the vascular injury response were studied inestrogen receptor α knockout (Lubahm et al., Proc. Nat'l Acad. Sci. USA90:11162, 1993) mice and their littermate controls using a cartoidinjury model. Vascular medial are increases and smooth muscle cellproliferation were quantitated 14 days following cartoid injury inovariectomized female mice treated with vehicle or physiologic levels of17-β estradiol. Suprisingly, 17-β estradiol markedly inhibited allmeasures of vascular injury both in wild-type and estrogen receptor αknockout mice to the same degree. These data suggest that estrogeninhibits vascular injury by a novel mechanism that is independent ofestrogen receptor α.

Use

The present invention also encompasses pharmaceutical compositions whichinclude a vasoprotective agent identified using the above-describedmethods. These compositions include a pharmaceutically effective amountof the vasoprotective agent in a pharmaceutically acceptable carrier ordiluent. Acceptable carriers or diluents for therapeutic use are wellknown in the pharmaceutical art, and are described, for example, inRemington's Pharmaceutical Sciences, Mack Publishing Co. (Gennaro ed.,1985). Preservatives, stabilizers, dyes and even flavoring agents may beprovided in the pharmaceutical composition. For example, sodiumbenzoate, sorbic acid and esters of p-hydroxybenzoic acid may be addedas preservatives. In addition, antioxidants and suspending agents may beused.

The compositions of the present invention may be formulated and used astablets, capsules or elixirs for oral administration, suppositories forrectal administration, sterile solutions, suspensions for injectableadministration, and the like. Injectables can be prepared inconventional forms, either as liquid solutions or suspensions, solidforms suitable for solution or suspension in liquid prior to injection,or as emulsions. Suitable excipients are, for example, water, saline,dextrose, mannitol, lactose, lecithin, albumin, sodium glutamate,cysteine hydrochloride, and the like. In addition, if desired, theinjectable pharmaceutical compositions may contain minor amounts ofnontoxic auxiliary substances, such as wetting agents, pH bufferingagents, and the like. If desired, absorption enhancing preparations(e.g., liposomes) may be utilized.

The pharmaceutically effective amount of the composition required as adose will depend on the route of administration, the type of animalbeing treated, and the physical characteristics of the specific animalunder consideration. The dose can be tailored to achieve optimalefficacy but will depend on such factors as weight, diet, concurrentmedication and other factors which those skilled in the medical artswill recognize. Typically, human clinical applications of products arecommenced at lower dosage levels, with dosage level being increaseduntil the desired effect is achieved. The determination of effectivedosage levels, that is the dosage levels necessary to achieve thedesired result, will be within the ambit of one skilled in the art basedon generally accepted protocols for clinical studies.

In practicing the methods of the invention, the vasoprotective agentscan be used alone or in combination with one another, or in combinationwith other therapeutic or diagnostic agents. A vasoprotective agent canbe administered parenterally, intravenously, subcutaneously,intramuscularly, colonically, rectally, nasally or intraperitoneally,employing a variety of dosage forms.

The smooth muscle cell line WB572 was deposited with the American TypeCulture Collection (ATCC), Rockville, Md., on Jul. 16, 1996 and givenaccession number CRL-12151.

The above-noted culture has been deposited under conditions that assurethat access to the culture will be available during the pendency of thepatent application to one determined by the Commissioner of Patents andTrademarks to be entitled thereto under 37 CFR 1.14 and 35 U.S.C. 122.The deposit is available as required by foreign patent laws in countrieswherein counterparts of the subject application, or its progeny, arefiled. However, it should be understood that the availability of thedeposit does not constitute a license to practice the subject inventionin derogation of patent rights granted by governmental action.

Further, the subject culture deposit will be stored and made availableto the public in accord with the provisions of the Budapest Treaty forthe Deposit of Microorganimms, i.e., it will be stored with all the carenecessary to keep them viable and uncontaminated for a period of atleast five years after the most recent request for the furnishing of asample of the deposit, and in any case, for a period of at least 30(thirty) years after the date of deposit of for the enforceable life ofany patent which may issue disclosing the culture plus five years afterthe last request for a sample from a deposit. The depositor acknowledgesthe duty to replace the deposit should the depository be unable tofurnish a sample when requested, due to the condition of the deposit.All restrictions on the availability to the public of the subjectculture deposits will be irrevocably removed upon the granting of apatent disclosing them.

What is claimed is:
 1. A method for evaluating whether an agent is acardiovascular therapeutic agent, comprising:(a) culturing vascularendothelial cells containing a non-endogenous reporter construct in thepresence and absence of a predetermined amount of said agent, saidnon-endogenous reporter construct comprising an estrogen responsiveelement operatively linked to a reporter gene; (b) culturingnon-vascular cells containing said non-endogenous reporter construct inthe presence and absence of said predetermined amount of said agent; and(c) measuring the expression of said reporter gene in said vascularendothelial cells in the presence and absence of said predeterminedamount of said agent and measuring the expression of said reporter genein said non-vascular cells in the presence and absence of saidpredetermined amount of said agent; whereby, said agent is identified asa cardiovascular therapeutic agent when the ratio of the expression ofsaid reporter gene in said vascular endothelial cells in the presence ofsaid predetermined amount of said agent to the expression of saidreporter in vascular endothelial cells in the absence of saidpredetermined amount of said agent is greater than the ratio of theexpression of said reporter gene in said non-vascular cells in thepresence of said predetermined amount of said agent to the expression ofsaid reporter gene in non-vascular cells in the absence of saidpredetermined amount of said agent.
 2. A method for evaluating whetheran agent is a cardiovascular therapeutic agent, comprising:(a) culturingvascular smooth muscle cells containing a non-endogenous reporterconstruct in the presence and absence of a predetermined amount of saidagent, said non-endogenous reporter construct comprising an estrogenresponsive element operatively linked to a reporter gene: (b) culturingnon-vascular cells containing said non-endogenous reporter construct inthe presence and absence of said predetermined amount of said agent; and(c) measuring the expression of said reporter gene in said vascularsmooth muscle cells in the presence and absence of said predeterminedamount of said agent and measuring the expression of said reporter genein said non-vascular cells in the presence and absence of saidpredetermined amount of said agent; whereby, said agent is identified asa cardiovascular therapeutic agent when the ratio of the expression ofsaid reporter gene in said vascular smooth muscle cells in the presenceof said predetermined amount of said agent to the expression of saidreporter in vascular smooth muscle cells in the absence of saidpredetermined amount of said agent is less than the ratio of theexpression of said reporter gene in said non-vascular cells in thepresence of said predetermined amount of said agent to the expression ofsaid reporter gene in non-vascular cells in the absence of saidpredetermined amount of said agent.
 3. A method for evaluating whetheran agent is a cardiovascular therapeutic agent, said methodcomprising:(a) culturing vascular endothelial cells containing anon-endogenous reporter construct in the presence and absence of apredetermined amount of said agent, said non-endogenous reporterconstruct comprising an estrogen responsive element operatively linkedto a reporter gene; (b) culturing non-estrogen receptor expressing cellscontaining said non-endogenous reporter construct in the presence andabsence of said predetermined amount of said agent; and (c) measuringthe expression of said reporter gene in said vascular endothelial cellsin the presence and absence of said predetermined amount of said agentand measuring the expression of said reporter gene in said non-estrogenreceptor expressing cells in the presence and absence of saidpredetermined amount of said agent; whereby, said agent is identified asa cardiovascular therapeutic agent when the ratio of the expression ofsaid reporter gene in said vascular endothelial cells in the presence ofsaid predetermined amount of said agent to the expression of saidreporter in vascular endothelial cells in the absence of saidpredetermined amount of said agent is greater than the ratio of theexpression of said reporter gene in said non-estrogen receptorexpressing cells in the presence of said predetermined amount of saidagent to the expression of said reporter gene in non-estrogen receptorexpressing cells in the absence of said predetermined amount of saidagent.
 4. A method for evaluating whether an agent is a cardiovasculartherapeutic agent, said method comprising:(a) culturing vascular smoothmuscle cells containing a non-endogenous reporter construct in thepresence and absence of a predetermined amount of said agent, saidnon-endogenous reporter construct comprising an estrogen responsiveelement operatively linked to a reporter gene; (b) culturingnon-estrogen receptor expressing cells containing said non-endogenousreporter construct in the presence and absence of said predeterminedamount of said agent; and (c) measuring the expression of said reportergene in said vascular smooth muscle cells in the presence and absence ofsaid predetermined amount of said agent and measuring the expression ofsaid reporter gene in said non-estrogen receptor expressing cells in thepresence and absence of said predetermined amount of said agent;whereby, said agent is identified as a cardiovascular therapeutic agentwhen the ratio of the expression of said reporter gene in said vascularsmooth muscle cells in the presence of said predetermined amount of saidagent to the expression of said reporter in vascular smooth muscle cellsin the absence of said predetermined amount of said agent is less thanthe ratio of the expression of said reporter gene in said non-estrogenexpressing cells cells in the presence of said predetermined amount ofsaid agent to the expression of said reporter gene in null cells in theabsence of said predetermined amount of said agent.
 5. A method forevaluating whether an amount is a cardiovascular therapeutic agent, saidmethod comprising:(a) culturing vascular endothelial cells containing anon-endogenous reporter construct in the presence and absence of apredetermined amount of said agent, said non-endogenous reporterconstruct comprising an estrogen responsive element operatively linkedto a reporter gene; (b) culturing vascular smooth muscle cellscontaining said non-endogenous reporter construct in the presence andabsence of said predetermined amount of said agent; and (c) measuringthe expression of said reporter gene in said vascular endothelial cellin the presence and absence of said predetermined amount of said agentand measuring the expression of said reporter gene in said vascularsmooth muscle cells in the presence and absence of said predeterminedamount of said agent; whereby, said agent is identified as acardiovascular therapeutic agent when the ratio of the expression ofsaid reporter gene in said vascular endothelial cells in the presence ofsaid predetermined amount of said agent to the expression of saidreporter in vascular endothelial cells in the absence of saidpredetermined amount of said agent is greater than the ratio of theexpression of said reporter gene in said vascular smooth muscle cells inthe presence of said predetermined amount of said agent to theexpression of said reporter gene in control smooth muscle cells in theabsence of said predetermined amount of said agent.
 6. The method ofclaim 4 wherein said vascular smooth muscle cells are WB572.
 7. Themethod of any of claims 1, 2, 3, 4, and 5 in which said cells are stablytransfected with said non-endogenous reporter construct.
 8. The methodof any of claims 1, 2, 3, 4, and 5 in which said cells areco-transfected with an estrogen receptor expression construct.
 9. Themethod of claim 8 wherein said estrogen receptor expression constructexpresses estrogen receptor α.
 10. The method of claim 8 wherein saidestrogen receptor expression construct expresses estrogen receptor β.